Modulation of Oncogenic Signaling in Glioblastomas

描述(申请人提供)：胶质瘤中致癌信号的调节。人类胶质母细胞瘤的一个特点是，在EGFR和EGFRvIII等过表达基因的刺激下，致癌信号异常活跃，使这些肿瘤高度恶性，并固有地对联合治疗产生抵抗，导致恶性胶质瘤患者预后极差。这一临床挑战的一个主要障碍是缺乏对这些肿瘤侵袭性背后的机制的完全了解，以及胶质瘤治疗的无效。本项目的目标是确定ESDN、内皮细胞和血管内皮细胞来源的神经粘连素样蛋白(又称DCBLD2和CLCP1)在恶性胶质母细胞瘤的发生和耐药中的作用。ESDN在血管损伤、转移性肺癌和乳腺癌中高表达。我们的初步研究表明，在约50%的临床胶质瘤标本中，ESDN基因被扩增，并且在过度表达EGFR和EGFRvIII的经典型和间叶型胶质母细胞瘤中优先上调。在肿瘤移植瘤中敲除ESDN显著抑制了EGFRvIII促进的动物脑内胶质瘤的生长和侵袭。在机制上，EGFRvIII和EGF诱导ESDN的Y621和Y750的酪氨酸磷酸化，激活TRAF6-Akt-和CrkII-rac1-信号，促进胶质母细胞瘤细胞的存活和侵袭。基于这些观察，在本项目中，我们提出了一项研究计划，旨在阐明ESDN促进EGFRvIII刺激的胶质瘤形成的机制，并探索靶向ESDN抑制EGFRvIII表达的脑胶质瘤的治疗应用。我们将首先研究ESDN如何通过TRAF6的解离和CrkII与ESDN的特定酪氨酸残基结合来影响EGFRvIII刺激的胶质瘤生长和侵袭，ESDN被EGF和EGFRvIII刺激而磷酸化。然后，我们将确定在原发胶质瘤标本中通过激活致癌的EGFR/EGFRvIII信号通路来诱导ESDN的p-Y的临床意义(目标1和2)。使用短期培养的原代胶质母细胞瘤(GBM)异种移植和诱导的新型脑胶质瘤模型，我们将探索临床前方法来评估抑制ESDN对化疗药物顺铂和替莫唑胺对动物脑内表达EGFR和EGFRvIII的胶质母细胞瘤的影响(目标3)。因此，这一提议揭示了一条未知的信号通路，即EGFR和EGFRvIII通过ESDN刺激TRAF6-Akt-和CrkII-信号，从而促进胶质瘤的生长和侵袭。阐明这一新的机制有望更好地理解致癌信号促进的胶质瘤发生，并克服胶质瘤对当前治疗方法的潜在耐药性。该项目还解决了神经肿瘤学中的一个紧迫挑战，并为开发可能被证明有效的新疗法提供了巨大的潜力，从而消除了治疗恶性胶质母细胞瘤患者的主要障碍。

DESCRIPTION (provided by applicant): Modulation of Oncogenic Signaling in Gliomas. A hallmark of human glioblastomas is that oncogenic signalings stimulated by overexpressed genes such as EGFR and EGFRvIII are aberrantly active, rendering these tumors highly malignant, and inherently resistant to combination therapies, resulting in an extremely poor prognosis of patients with malignant gliomas. A major barrier of this clinical challenge is the lack of a complete understanding of the mechanisms underlying the aggressiveness of these tumors and ineffectiveness of therapies for gliomas. The goal of this project is to define the role of ESDN, endothelial and smooth muscle cell-derived neuropilin-like protein (other names, DCBLD2 and CLCP1) in tumorigenesis and therapy-resistance of malignant human glioblastomas. ESDN is expressed at high levels in vascular injury, metastatic lung cancer and breast cancer. Our preliminary studies show that the ESDN gene is amplified in ~50% of clinical glioma specimens and preferentially up-regulated in classical and mesenchymal subtypes of glioblastomas that overexpress EGFR and EGFRvIII. Knockdown of ESDN in tumor xenografts significantly suppressed EGFRvIII-promoted glioma growth and invasion in the brain of animals. Mechanistically, EGFRvIII and EGF induce tyrosine phosphorylation (p-Y) of Y621 and Y750 of ESDN that activate TRAF6-Akt- and CrkII-Rac1- signaling and promote glioblastoma cell survival and invasion. Based on these observations, in this project, we propose a research plan directed at elucidating mechanisms by which ESDN augments EGFRvIII-stimulated glioma tumorigenesis and exploring therapeutic application of targeting ESDN to inhibit EGFRvIII-expressing brain gliomas. We will first investigate how ESDN affects EGFRvIII-stimulated glioma growth and invasion through dissociation of TRAF6 and binding of CrkII to specific tyrosine residues of ESDN that are phosphorylated by EGF and EGFRvIII stimulation. We will then determine clinical significance of the induced p-Y of ESDN with activation of oncogenic EGFR/EGFRvIII signaling pathways in primary glioma tumor specimens (Aims 1 and 2). Using short-term cultured primary glioblastoma (GBM) xenograft and an induced de novel brain glioma models, we will explore pre-clinical approaches to assess the impact of inhibition of ESDN on the efficacies of chemotherapeutic agents, cisplatin and temozolomide on EGFR- and EGFRvIII-expressing glioblastomas in the brain of animals (Aim 3). Therefore, this proposal reveals an unrecognized signaling path by which EGFR and EGFRvIII stimulate TRAF6-Akt- and CrkII-signaling through ESDN, thereby promoting glioma growth and invasion. Elucidation of this novel mechanism holds promise to a better understanding of the oncogenic signaling-promoted glioma tumorigenesis and to overcome the insidious glioma resistance to current therapies. This project also addresses an urgent challenge in neuro-oncology and offers an enormous potential for developing novel therapies that could prove effective, thus eliminating the major barrier in treating patients with malignant glioblastomas.